This invention relates to a process for isolating a stable form of nerve growth factor (NGF) and to the stable NGF obtained.
At the present time, three different molecular forms of NGF have been recognized. Two of these have been isolated from male mouse submandibular glands and have been studied extensively. A third type of NGF is that secreted by mouse L cells in culture. One submandibular gland form has been called 2.5S NGF. This species is a dimer (molecular weight 26,000) composed of two noncovalently linked, identical polypeptide chains whose primary structures are known. In dilute solution, the dimer dissociates into its monomeric subunits (molecular weight 13,000), which are the biologically active species in stimulating ganglionic neurite outgrowth in vitro. The other submandibular gland form has been named 7S NGF. This protein is isolated and purified from submandibular glands by a different procedure from that used to obtain 2.5S NGF. It has a molecular weight of about 140,000 and is composed of three different proteins termed .alpha., .beta. and .gamma.. The .alpha. and .gamma. constituents have no known biological function, although the .gamma. species displays arginine esterase activity. Only the .beta. component of 7S NGF is biologically active in stimulating neurite outgrowth; by biological and immunological criteria, .beta.-NGF is indistinguishable from the 2.5S NGF. These two species differ only in that during isolation of either one, limited and different proteolytic modifications occur, the extent of which depends upon the isolation conditions.
The NGF secreted by L cells is yet another species. This protein has a molecular weight close to 160,000 and, like 7S NGF, it contains 2.5S NGF as part of its structure. However, L-cell NGF differs from the 7S complex in at least one important respect. The 7S NGF structure is unstable in solution at neutral pH, and dissociates completely to yield a mixture of its components at concentrations (.mu.g/ml) nearly 1000 times higher than those required to display biological activity (about 1 ng/ml). Consequently, the biological activity of 7S NGF is due entirely to its 2.5S (.beta.) component. In contrast to the marked instability of 7S NGF, L-cell NGF is completely stable in very dilute solution and does not dissociate even at concentrations as low as 1 ng/ml. This appreciable difference in stability between gland and L-cell NGF is surprising, since both proteins are of mouse origin. Yet the mouse submandibular gland is rich in proteases, and it seems possible that proteolysis occurring during the process of purification of 7S NGF could account for the instability of the protein.
Prior to the present invention, the known forms of NGF have had no known biological activity other than the stimulation of ganglionic neurite outgrowth.